The present disclosure, in some embodiments thereof, relates to isolated dsRNA molecules and methods of using same for silencing target molecules of interest.
RNA interference (RNAi) has been shown effective in silencing gene expression in a broad variety of species, including plants, with wide ranging implications for cancer, inherited disease, infectious disease in plants and animals. Studies in a variety of organisms have shown that effectors of RNAi include dsRNA and related small interfering RNAs (siRNAs; also called “short interfering RNAs” and “silencing RNAs”). Studies have also shown in a variety of organisms that dsRNA or their siRNA derivatives can be used to arrest, retard or even prevent a variety of pathogens, most notably viral diseases (see, for example, PCT Patent Application Publication No. WO/2003/004649).
It has been shown in some species that RNAi mediated interference spreads from the initial site of dsRNA delivery, producing interference phenotypes throughout the injected animal. Recently the same spreading effect of dsRNA has been demonstrated in bee larva. In addition, homologs of transmembrane proteins called systemic RNA interference defective proteins (SID) have been detected in, for example, humans, mouse and C. elegans. It is thought that SID transmembrane channels are responsible for endocytic uptake and spreading effect of dsRNA (Aronstein et al., J. Apic Res and Bee World, 2006; 45:20-24; see also van Roessel P, Brand A H., “Spreading silence with Sid,” Genome Biol. 5(2):208 (2004)).
Application of RNA interference technology for insects that are plant pests and other plant pests has been suggested. Moderate RNAi-type silencing of insect genes by feeding has been demonstrated (Turner et al., Insect Mol Biol 2006; 15:383; and Araujo et al., Insect Mol. Biol. 2006; 36:683). Various publications have since then focused on the incorporation of dsRNA in plants as pesticides. Such incorporation methods can be divided into transgenic gene expression and coating such as a seed coating.
U.S. Pat. No. 6,326,193 refers to the use of recombinant insect viruses such as baculoviruses expressing dsRNA to silence selected insect genes for pest control. PCT Patent Application Publication No. WO 99/32619 describes the use of dsRNA for reducing crop destruction by plant pathogens or pests such as arachnids, insects, nematodes, protozoans, bacteria, or fungi. PCT Patent Application Publication No. WO 2004/005485 describes RNAi sequences and transgenic plants designed to control plant-parasitic nematodes.
U.S. Patent Application Publication No. 20030154508 describes pest control with a dsRNA against a cation-amino acid transporter/channel protein. PCT Patent Application Publication No. WO 02/14472 describes an inverted repeat and a sense or antisense nucleic acids for inhibiting target gene expression in a sucking insect. U.S. Patent Application Publication No. 20030150017 describes the use of RNA molecules homologous or complementary to a nucleotide sequence of a plant pest such as nematodes and insects.
Raemakers et al. (PCT Patent Application Publication Nos. WO 2007/080127 and WO 2007/080126) have disclosed transgenic plants expressing RNAi for controlling pest infestation by insects, nematodes, fungus and other plant pests. Among the sequences taught are sequences targeting essential genes of insects. Waterhouse et al. (U.S. Patent Application Publication No. 20060272049) and Van De Craen (U.S. Patent Application Publication No. 2010068172) also disclosed transgenic plants expressing dsRNA directed to essential genes of plant insect pests, for use as pesticides and insecticides. Boukharov et al. (U.S. Patent Application Publication No. 20070250947) disclosed dsRNA in transgenic plants for targeting plant parasitic nematodes.
U.S. Patent Application Publication No. 20080022423 describes the control of fungal and oomycete plant pathogens by inhibiting one or more biological functions. The disclosure provides methods and compositions for such control. By feeding one or more recombinant double stranded RNA molecules provided by the disclosure to the pathogen, a reduction in disease may be obtained through suppression of gene expression. The disclosure is also directed to methods for making transgenic plants that express the double stranded RNA molecules, and to particular combinations of transgenic agents for use in protecting plants from pathogen infection. Also described is a seed coating with the dsRNA anti-pathogenic compositions.
PCT Patent Application Publication No. WO 2011112570 describes a method of regulating target endogenous gene expression in growing plants/plant organs involving topically coating onto plants/organs, a composition comprising polynucleotide having sequence of specific contiguous nucleotides, and a transferring agent.
U.S. Pat. No. 8,143,480 refers to methods for knock-down of a target genes in plants, particularly efficient and specific methods for knock-down of a target gene in plants. This disclosure also relates to methods for silencing endogenous plant genes or plant pathogen genes. It further relates to nucleic acid constructs (DNA, RNA) which comprise a nucleic acid sequence that corresponds to a target gene or fragment thereof flanked by two complementary sites to an smRNA, e.g., a miRNA (one complementary site is on either side of the nucleic acid sequence), resulting in, for example the configuration: complementary site—nucleic acid sequence that corresponds to a target gene—complementary site. Axtell and Bartell describe siRNA biogenesis in Arabidopsis (Axtell and Bartel Cell. 2006 Nov. 3; 127(3):565-77.).
It has been reported that an autonomous dsRNA sequence derived from endovirus is found in every tissue of an infected plant and at every developmental stage. Thus, in 1993 Fukuhara et al. (Plant Mol. Biol. 21(6):1121-1130) identified a linear, 16 kb, dsRNA in symptomless Japonica rice that is not found in Indica rice. The dsRNA was detected in every tissue and at every developmental stage and its copy number was approximately constant (about 20 copies/cell). A sequence of about 13.2 kb of the dsRNA was determined and two open reading frames (ORFs) were found. The larger ORF (ORF B) was more than 12,351 nucleotides long and encoded a polypeptide of more than 4,117 amino acid residues having an RNA helicase-like domain followed by an RNA dependent RNA Polymerase-like domain, as characterized in subsequent works published as Fukuhara et al. 1995 J. Biol. Chem. 270(30):18147-18149; and Moriyama et al. 1995 Mol. Gen. Genet. 248(3):364-369.
While not limited by theory, during RNA silencing, RNAs of about 21 to 24 nucleotides (nt) in length are generated, which are incorporated into a protein complex where they serve as guide RNAs to direct the down-regulation of gene expression at the transcriptional or posttranscriptional level. These small interfering RNAs, small silencing RNAs, or short interfering RNAs are called “siRNAs” or “microRNAs”, depending upon their biogenesis: endogenous siRNAs derive from long double-stranded RNA and miRNAs derive from local hairpin structures within longer transcripts.
RNA silencing occurs in plants, insects, nematodes and other animals. In addition, new compositions (e.g., nucleic acid constructs) and methods of achieving RNA-based silencing would be useful, and plants in which expression of one or more genes of interest is modulated, e.g., inhibited, would be of great use. New compositions and rapid cost-effective methods of achieving RNA-based silencing by directly manipulating the plant seed are highly desirable.